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Engineering intracellular biomineralization and biosensing by a magnetic protein.

ABSTRACT: Remote measurement and manipulation of biological systems can be achieved using magnetic techniques, but a missing link is the availability of highly magnetic handles on cellular or molecular function. Here we address this need by using high-throughput genetic screening in yeast to select variants of the iron storage ferritin (Ft) that display enhanced iron accumulation under physiological conditions. Expression of Ft mutants selected from a library of 10(7) variants induces threefold greater cellular iron loading than mammalian heavy chain Ft, over fivefold higher contrast in magnetic resonance imaging, and robust retention on magnetic separation columns. Mechanistic studies of mutant Ft proteins indicate that improved magnetism arises in part from increased iron oxide nucleation efficiency. Molecular-level iron loading in engineered Ft enables detection of individual particles inside cells and facilitates creation of Ft-based intracellular magnetic devices. We demonstrate construction of a magnetic sensor actuated by gene expression in yeast.

SUBMITTER: Matsumoto Y

PROVIDER: S-EPMC4667635 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Engineering intracellular biomineralization and biosensing by a magnetic protein.

Matsumoto Yuri Y Chen Ritchie R Anikeeva Polina P Jasanoff Alan A

Nature communications 20151102

Remote measurement and manipulation of biological systems can be achieved using magnetic techniques, but a missing link is the availability of highly magnetic handles on cellular or molecular function. Here we address this need by using high-throughput genetic screening in yeast to select variants of the iron storage ferritin (Ft) that display enhanced iron accumulation under physiological conditions. Expression of Ft mutants selected from a library of 10(7) variants induces threefold greater ce ...[more]

PMID: 26522873

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Project description:BackgroundMembrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved.ResultsWe engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER.ConclusionsWe conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.

Dataset Information

Engineering intracellular biomineralization and biosensing by a magnetic protein.

Publications

Engineering intracellular biomineralization and biosensing by a magnetic protein.

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